Transcriptional enhancers enable exquisite spatiotemporal control of gene expression in metazoans. Enrichment of monomethylation of histone H3 lysine 4 (H3K4me1) is a major chromatin signature of transcriptional enhancers. Lysine (K)-specific demethylase 1A (KDM1A, also known as LSD1), an H3K4me2/me1 demethylase, inactivates stem-cell enhancers during the differentiation of mouse embryonic stem cells (mESCs). However, its role in undifferentiated mESCs remains obscure. Here, we show that KDM1A actively maintains the optimal enhancer status in both undifferentiated and lineage-committed cells. KDM1A occupies a majority of enhancers in undifferentiated mESCs. KDM1A levels at enhancers exhibit clear positive correlations with its substrate H3K4me2, H3K27ac, and transcription at enhancers. In Kdm1a-deficient mESCs, a large fraction of these enhancers gains additional H3K4 methylation, which is accompanied by increases in H3K27 acetylation and increased expression of both enhancer RNAs (eRNAs) and target genes. In postmitotic neurons, loss of KDM1A leads to premature activation of neuronal activity-dependent enhancers and genes. Taken together, these results suggest that KDM1A is a versatile regulator of enhancers and acts as a rheostat to maintain optimal enhancer activity by counterbalancing H3K4 methylation at enhancers.

Different ancestral autosomes independently evolved into sex chromosomes in snakes, birds, and mammals. In snakes and birds, females are ZW and males are ZZ; in mammals, females are XX and males are XY. Although X and Z Chromosomes retain nearly all ancestral genes, sex-specific W and Y Chromosomes suffered extensive genetic decay. In both birds and mammals, the genes that survived on sex-specific chromosomes are enriched for broadly expressed, dosage-sensitive regulators of gene expression, subject to strong purifying selection. To gain deeper insight into the processes that govern survival on sex-specific chromosomes, we carried out a meta-analysis of survival across 41 species-three snakes, 24 birds, and 14 mammals-doubling the number of ancestral genes under investigation and increasing our power to detect enrichments among survivors relative to nonsurvivors. Of 2564 ancestral genes, representing an eighth of the ancestral amniote genome, only 324 survive on present-day sex-specific chromosomes. Survivors are enriched for dosage-sensitive developmental processes, particularly development of neural crest-derived structures, such as the face. However, there was no enrichment for expression in sex-specific tissues, involvement in sex determination or gonadogenesis pathways, or conserved sex-biased expression. Broad expression and dosage sensitivity contributed independently to gene survival, suggesting that pleiotropy imposes additional constraints on the evolution of dosage compensation. We propose that maintaining the viability of the heterogametic sex drove gene survival on amniote sex-specific chromosomes, and that subtle modulation of the expression of survivor genes and their autosomal orthologs has disproportionately large effects on development and disease.

Recent evidence from proteomics and deep massively parallel sequencing studies have revealed that eukaryotic genomes contain substantial numbers of as-yet-uncharacterized open reading frames (ORFs). We define these uncharacterized ORFs as novel ORFs (nORFs). nORFs in humans are mostly under 100 codons and are found in diverse regions of the genome, including in long noncoding RNAs, pseudogenes, 3' UTRs, 5' UTRs, and alternative reading frames of canonical protein coding exons. There is therefore a pressing need to evaluate the potential functional importance of these unannotated transcripts and proteins in biological pathways and human disease on a larger scale, rather than one at a time. In this study, we outline the creation of a valuable nORFs data set with experimental evidence of translation for the community, use measures of heritability and selection that reveal signals for functional importance, and show the potential implications for functional interpretation of genetic variants in nORFs. Our results indicate that some variants that were previously classified as being benign or of uncertain significance may have to be reinterpreted.

The predominant methodology for DNA methylation analysis relies on the chemical deamination by sodium bisulfite of unmodified cytosine to uracil to permit the differential readout of methylated cytosines. Bisulfite treatment damages the DNA, leading to fragmentation and loss of long-range methylation information. To overcome this limitation of bisulfite-treated DNA, we applied a new enzymatic deamination approach, termed enzymatic methyl-seq (EM-seq), to long-range sequencing technologies. Our methodology, named long-read enzymatic modification sequencing (LR-EM-seq), preserves the integrity of DNA, allowing long-range methylation profiling of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) over multikilobase length of genomic DNA. When applied to known differentially methylated regions (DMRs), LR-EM-seq achieves phasing of >5 kb, resulting in broader and better defined DMRs compared with that previously reported. This result showed the importance of phasing methylation for biologically relevant questions and the applicability of LR-EM-seq for long-range epigenetic analysis at single-molecule and single-nucleotide resolution.

Within-species contamination is a major issue in sequencing studies, especially for mitochondrial studies. Contamination can be detected by analyzing the nuclear genome or by inspecting polymorphic sites in the mitochondrial genome (mtDNA). Existing methods using the nuclear genome are computationally expensive, and no appropriate tool for detecting sample contamination in large-scale mtDNA data sets is available. Here we present haplocheck, a tool that requires only the mtDNA to detect contamination in both targeted mitochondrial and whole-genome sequencing studies. Our in silico simulations and amplicon mixture experiments indicate that haplocheck detects mtDNA contamination accurately and is independent of the phylogenetic distance within a sample mixture. By applying haplocheck to The 1000 Genomes Project Consortium data, we further evaluate the application of haplocheck as a fast proxy tool for nDNA-based contamination detection using the mtDNA and identify the mitochondrial copy number within a mixture as a critical component for the overall accuracy. The haplocheck tool is available both as a command-line tool and as a cloud web service producing interactive reports that facilitates the navigation through the phylogeny of contaminated samples.

Our core timekeeping mechanism, the circadian clock, plays a vital role in immunity. Although the mechanics of circadian control over the immune response is generally explained by transcriptional activation or repression derived from this clock's transcription-translation negative-feedback loop, research suggests that some regulation occurs beyond transcriptional activity. We comprehensively profiled the transcriptome and proteome of murine bone marrow-derived macrophages and found that only 15% of the circadian proteome had corresponding oscillating mRNA, suggesting post-transcriptional regulation influences macrophage clock regulatory output to a greater extent than any other tissue previously profiled. This regulation may be explained by the robust temporal enrichment we identified for proteins involved in degradation and translation. Extensive post-transcriptional temporal-gating of metabolic pathways was also observed and further corresponded with daily variations in ATP production, mitochondrial morphology, and phagocytosis. The disruption of this circadian post-transcriptional metabolic regulation impaired immune functionality. Our results demonstrate that cell-intrinsic post-transcriptional regulation is a primary driver of circadian output in macrophages and that this regulation, particularly of metabolic pathways, plays an important role in determining their response to immune stimuli.